Project description:Transthyretin-derived amyloidosis (ATTR) is a degenerative, systemic disease characterized by transthyretin fibril deposition in organs like the heart, kidneys, liver, and skin. We report the first cryo-EM structure of transthyretin fibrils isolated from skin tissue of a living patient with a rare genetic mutation (ATTRv F64S). The structure adopts a highly conserved fold previously observed in other ATTR fibrils from different tissues or genetic variants. Mass spectrometry was used to identify common post-translational modifications. The structural consistency between ATTR filaments validates non-invasive skin biopsy as a diagnostic tool.
Project description:The microtubule associated protein Tau (MAPT) expressed in neurons is involved in microtubules stabilization, cell morphogenesis and axonal transport. In pathological conditions, Tau assembles into high molecular weight assemblies leading to neuropathological Tau deposits, the hallmark of several neurodegenerative diseases collectively named “tauopathies”, including Alzheimer’s disease. These pathologic Tau assemblies are released by affected neuronal cells and taken up by naïve neighbor cells, propagate from one cell to another and amplify by seeding the aggregation of endogenous Tau. In order to identify plasma membrane proteins exposed extracellularly that interact with extracellularly applied fibrillar Tau assemblies, we exposed pure-neuronal cultures to fibrillar Tau for 10 min, pulled down the associated proteins, and identified them using a proteomic-based approach. Of the six Tau isoforms produced by alternative splicing of the MAPT gene and differing from each other by the presence or absence of one or two inserts in the N-terminal part (0N, 1N or 2N) of the protein and by the presence of either three or four repeated microtubule binding motifs in the protein C-terminal part (3R or 4R), we have expressed and purified 1N3R and 1N4R Tau isoforms and further assembled them into 1N3R and 1N4R Tau fibrils. Using pull-down of whole cell lysates and mass spectrometry, we have identified proteins interacting with extracellularly applied Tau fibrils. We have performed two different experiments with either 1N3R Tau fibrils or 1N4R Tau fibrils (condition 1 and condition 2 respectively). Each condition consists in six experimental replicates of cells exposed 10 min to Tau fibrils and of the non-treated cells used as controls.
Project description:Mimivirus 1.2Mb genome is organized into a 30 nm nucleocapsid-like structure made of two closely related GMC-oxidoreductases, also composing the fibrils decorating its virions. In this work, we used MS-proteomics to characterize the protein content of virions and fibrils from different members of the Mimiviridae family (clade A: Mimivirus reunion -Mr- and Mimivirus M4 -M4, clade B: Moumouvirus australiensis -Ma- and Moumouvirus maliensis -Mm, clade C: Megavirus chilensis -Mc- and Megavirus vitis -Mv). Furthermore, we analyzed fractions purified from Mr mutants devoid of one of the two GMC-oxidoreductases (Mr_KOqu_143 and Mr_KOqu_946), or of both GMC-oxidoreductases (Mr_2KO) with or without expression of the GFP fused to the N-terminus of one GMC-oxidoreductase (Mr_2KO-GFP). Our results show the versatility of the protein content of the fibrils, with fibrils composed of different proteins inter- and even intra-clade, clades B and C viruses presenting fibrils with a protein composition closer to each other than that of clade A viruses.
Project description:The calcifying matrix proteins embedded within the shell biomineral structure is a mixture of proteins, glycoproteins, and polysaccharides that are involved in the shell formation processes. Here with develop bottom-up shotgun proteomic investigation of these proteins, thanks to specific transcriptomic data generated from the calcifying mantle of adult Lymnaea.
Project description:The structural characterization of pathogenic tau filaments that accumulate in tauopathies, including Alzheimer disease, is key for understanding their pathogenic function and identify the structural determinants of distinct tau filaments involved in distinct tauopathies. Fibrillar tau surfaces play a crucial role in tau fibrils interaction with various protein partners and binding to neurons, a key step for their prion-like propagation propensity. In order to identify the amino acid residues stretches that are exposed and those that are not exposed to the solvent within tau fibrils, we used structural proteomic approaches: proteolysis and molecular covalent surface painting using NHS-Biotin followed by MS-based analysis of the proteolytic products. We compared the solvent accessible amino acid residues on the surface of htau proteins upon assembly of 1N3R and 1N4R human htau monomers into fibrils in vitro. We mapped the amino acid segments exposed or unexposed on the surface of htau fibrils by determining the accessibility GluC enzyme and NHS-biotin followed by proteolysis. The proteolytic peptides were analyzed by nanoLC-MSMS and processed using Mascot for their identification and PeakView for intensity extraction of labeled and unlabeled proteolytic peptides.
Project description:Background: An important feature to vitamin D physiology is its gender dependence. The aim of this study was to examine whether vitamin D exerts a sexually dimorphic effect on the blood coagulation pathway among adults with overweight. Methods: This study compared the serum proteomic profiles of age and BMI-matched males (n=26) and pre-menopausal females (n=24) with overweight that attained vitamin D sufficiency after a 12-month intervention. Unprocessed serum was subjected to depletion-free, quantitative proteomic analysis using our previously published methodology. Results. A total of 1,841 proteins were profiled (p < 0.05). The analysed proteins vitamin-K dependent protein C, von Willebrand factor, fibrinogen gamma chain and multimerin-1 were ELISA validated to be differentially affected between genders by vitamin D status improvement. Conclusions: Vitamin D optimization exhibits a sexually dimorphic effect on the blood coagulation pathway among adults with overweight. This gender specific vitamin D effect should be taken into consideration in the design and interpretation of vitamin D observational and intervention studies.
Project description:The surface associated proteomics of different P aeruginosa fap genetic variants were evaluated. The bacteria were grown on the glass wool, the biomass was extracted from glass wool and the surface influenced planktonics(SIP)biomass was collected.
Project description:To characterise the abundance of ubiquitylated Tau peptides in the presence of active or inactive variants (wt 196-565 vs. C221A) of the broad spectrum deubiquitylase USP21. Tau peptides are obtained from Tau fibrils purified from post mortem human brains diagnosed with Alzheimer’s. Their detection is facilitated by previous training of the mass spectrometer with in vitro ubiquitylated Tau. The same samples were used to evaluate the percentage of peptides modified with ubiquitin
Project description:Ancient proteins were extracted from ceramic vessels from the West Mound of the key early farming site of Çatalhöyük, dating to the early 6th millennium BC. This analysis revealed that barley, wheat, pulses, as well as goat, sheep and bovinae dairy and meat products were contained in ceramic vessels. Moreover, this demonstrates that dietary proteins can be identified from archaeological artefacts from up to 6,000 BC and that this approach can unlock detailed evidence of past culinary practices, with more taxonomic and tissue-specific clarity than has been possible with previous biomolecular techniques.
Project description:SPINT1 is a membrane-anchored serine protease inhibitor that modulates pericellular proteolysis. The pancreas-specific disruption of Spint1 in mice significantly decreased islet size and mass, leading to glucose intolerance, which was causally related to the down-regulation of MafA and insulin. Mechanistically, the silencing of Hepsin (encoding a SPINT1 target protease) counteracted the repressive effect of Spint1 knockdown on MafA and Ins1 expression in β cells. Together, the results suggest that SPINT1 plays a novel role in β cells to maintain glucose homeostasis and insulin production.